Method of multiplex immune profiling of mouse blood cells with highly sensitive detection of reporter β-galactosidase LacZ

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Bacterial β-galactosidase (LacZ) has been widely used as a reporter in the creation of mouse lines to study gene expression. However, LacZ reporters have limitations related to the presence of endogenous β-galactosidase in cells, as well as the low sensitivity and penetrating ability of existing substrates to detect LacZ activity. Multicolor flow cytometry analysis of gene expression in living cells requires precise, sensitive, non-toxic fluorescent indicators. In this study, we evaluated the effectiveness of the immobilized SPiDER-βGal fluorescent probe for LacZ detection in main populations of blood cells of reporter mice by multicolored flow cytometry. The results showed that SPiDER-βGal was highly sensitive to LacZ, but it also detected endogenous β-galactosidase. Myeloid cells had the highest background activity. Application of the proton pump inhibitor Bafilomycin A1 elevates lysosomal pH and increases the resolution of LacZ detection in leukocyte populations by suppressing background endogenous β-galactosidase activity. Extending the incubation with the SPiDER-βGal to 60 minutes improved the sensitivity of the method tenfold. Thus, the use of specific inhibitors of lysosomal proton transport increases the resolution of LacZ activity analysis in reporter animals for multi-channel sorting of LacZ-expressing live leukocytes in the context of surface markers for further functional and genetic studies of blood populations.

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作者简介

V. Mihailovskaya

Sirius University of Science and Technology, Scientific Center for Genetics and Life Sciences

Email: rybtsov.sa@talantiuspeh.ru
俄罗斯联邦, 354340 Sirius

D. Bogdanova

Sirius University of Science and Technology, Scientific Center for Genetics and Life Sciences; Institute of Cytology, Russian Academy of Sciences

Email: rybtsov.sa@talantiuspeh.ru
俄罗斯联邦, 354340 Sirius; Institute of Cytology, Russian Academy of Sciences

O. Demidov

Sirius University of Science and Technology, Scientific Center for Genetics and Life Sciences; Institute of Cytology, Russian Academy of Sciences

Email: rybtsov.sa@talantiuspeh.ru
俄罗斯联邦, 354340 Sirius; 194064 St. Petersburg

S. Rybtsov

Sirius University of Science and Technology, Scientific Center for Genetics and Life Sciences

编辑信件的主要联系方式.
Email: rybtsov.sa@talantiuspeh.ru
俄罗斯联邦, 354340 Sirius

参考

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2. Fig. 1. Gating strategy for mouse peripheral blood cells

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3. Fig. 2. The studied populations of mouse blood leukocytes. a – t-SNE scatterplot with manually superimposed leukocyte populations (t-SNE iterations = 1000 and k = 30). b – Colored t-SNE scatterplots showing the expression level and distribution of the main markers

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4. Fig. 3. Comparison of fluorescent substrates GlycoGREEN (G) and SPiDER-βGal (S) for detection of LacZ activity in peripheral blood cell populations of C57Bl/6LacZ/LacZ (LacZ) and wild-type C57Bl/6 (WT) reporter mice. Shown are the fluorescence intensity of GlycoGREEN and SPiDER-βGal substrates (ΔMFI), * p < 0.05, *** p < 0.0005, ns – differences are statistically insignificant

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5. Fig. 4. Comparison of protocols for detection of LacZ activity in peripheral blood cells of reporter mice. Colored t-SNE scatterplots are shown showing fluorescence intensity for GlycoGREEN, SPiDER-βGal, and SPiDER-βGal substrates in combination with Bafilomycin A1, as well as fluorescence distribution across blood cell populations of wild-type C57Bl/6 (WT) and reporter C57Bl/6LacZ/LacZ (LacZ) mice.

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6. Fig. 5. SPiDER-βGal fluorescence intensity (ΔMFI) values ​​in different blood cell populations using SPiDER-βGal alone (S) and in combination with Bafilomycin A1 (SB). * p < 0.05, *** p < 0.0005, ns – differences are statistically insignificant

 


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7. Fig. 6. Distribution of blood cell populations by fluorescence intensity of SPiDER-βGal in combination with Bafilomycin A1 (SB) in wild-type C57Bl/6 (left) and transgenic C57Bl/6LacZ/LacZ mice (right). * p < 0.05, ** p < 0.005, *** p < 0.0005

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8. Fig. 7. Comparison of the effects of staining time of peripheral blood cells from wild-type C57Bl/6 (WT), heterozygous C57Bl/6LacZ/+ (LacZ+/−), and homozygous C57Bl/6LacZ/LacZ (LacZ+/+) reporter mice with SPiDER-βGal in combination with Bafilomycin A1. Shown is the SPiDER-βGal fluorescence intensity (ΔMFI), * p < 0.05, ** p < 0.005, *** p < 0.0005, ns – differences are not statistically significant

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9. Appendix
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